Regulation Of GTP-binding Proteins

adp核糖基化因子（ARFs）是gtp结合蛋白，可调节多种囊泡运输途径。ARF功能需要在gtp结合的活性形式和gdp结合的非活性形式之间进行调节。GTP的结合是由鸟嘌呤核苷酸交换蛋白（GEPs）催化的，其中一些是由brefeldin A （BFA）抑制的，brefeldin A是一种抑制蛋白质分泌并导致高尔基池可逆分解的药物。该小组较早地纯化和克隆了两个bfa抑制的GEPs (BIG1和BIG2)，它们通常在具有特异性抗体的培养细胞中一起沉淀，但在酵母双杂交筛选中，BIG1与FKBP13相互作用，BIG2与riα相互作用，riα是环amp活化蛋白激酶（PKA）的调节亚基。FKBP13是fk506结合蛋白家族中的一种亲免疫蛋白，不结合或抑制钙调磷酸酶。用FK506孵育Jurkat细胞，导致FKBP13和BIG1的核聚集，但它们没有共定位。此外，在无血清的细胞中，BIG1进入细胞核，在核基质和核仁中被证实，并与核孔蛋白相关。细胞核中与BIG1相关的rna的表征在接替者到来之前因博士后的意外离开而中断后仍在继续。

ADP-ribosylation factors (ARFs) are GTP-binding proteins that regulate numerous vesicular trafficking pathways. ARF function requires the regulated alternation between GTP-bound active and GDP-bound inactive forms. GTP binding is catalyzed by guanine nucleotide-exchange proteins (GEPs), some of which are inhibited by brefeldin A (BFA), a drug that inhibits protein secretion and causes reversible disintegration of Golgi cisternae. Two BFA-inhibited GEPs (BIG1 and BIG2) that were purified and cloned earler by the group often precipitated together from cultured cells with specific antibodies, but they behaved quite differently in yeast two-hybrid screens, where BIG1 interacted with FKBP13 and BIG2 with RIalpha, a regulatory subunit of cyclic AMP-activated protein kinase (PKA). FKBP13 is an immunophilin of the FK506-binding protein family that does not bind or inhibit calcineurin. Incubation of Jurkat cells with FK506 resulted in nuclear accumulation of both FKBP13 and BIG1, but they were not colocalized. In addition, BIG1 moved into nuclei in cells deprived of serum, where it was demonstrated in nuclear matrix and nucleoli as well as associated with nucleoporin. Characterization of RNAs associated with BIG1 in nuclei is continuing after interruption caused by unexpected departure of the responsible post-doctoral fellow before arrival of his replacement. Investigation of the RIalpha-BIG2 interaction led to identification of sequences corresponding to those in A kinase-anchoring proteins in the BIG2 molecules, as reported last year. A manuscript in preparation characterizes protein kinase A-dependent effects of cyclic AMP on nuclear accumulation of endogenous BIG1 and other proteins. Exploring the interaction of BIG2 with exocyst protein Exo70 (detected in the yeast two-hybrid system) led to demonstration of the two proteins in trans-Golgi network and centrosomes, as well as in exocyst structures or complexes that move along microtubules to the plasma membrane. All findings are consistent with their functional association in both early and late stages of vesicular transport.